Hybrid Tissue Scaffold For Tissue Engineering

ABSTRACT

A hybrid tissue scaffold is provided which comprises a porous primary scaffold having a plurality of pores and a porous secondary scaffold having a plurality of pores, wherein the secondary scaffold resides in the pores of the primary scaffold to provide a hybrid scaffold. The pores of the porous primary scaffold may have a pore size in a range of 0.50 mm to 5.0 mm, and the pores of the porous secondary scaffold may have a pore size in a range of 50 μm to 600 μm. The primary scaffold may provide 5% to 30% of a volume of the hybrid scaffold.

FIELD OF THE INVENTION

The present disclosure relates to a hybrid tissue scaffold for tissueimplants and methods of for use thereof. The hybrid tissue scaffoldincludes a primary three-dimensional (base) scaffold which supports orotherwise carries a secondary three-dimensional scaffold, particularlywithin the pores thereof.

BACKGROUND

Engineered tissue implants require a tissue scaffold that is preferablymade of similar materials to the tissue that will be replaced by theengineered tissue implant. This requires using biomaterials such as, butnot limited to, collagen, fibrin, and glycosaminoglycans (GAGs).However, while these materials may provide the correct biomaterialenvironment, they lack the mechanical strength required to be implanted.Such forces may be generated from in situ forces, such as pressure fromfluids passing through or by the implant. Further, the tissue implantsneed to be able to withstand the forces of being handled and surgicallyimplanted by the surgeon. This is a common, long standing problem thathas made designing a tissue engineering implant very difficult.

Many different techniques have been used to try to overcome the lack ofmechanical strength in scaffolds, and the corresponding lack of strengthin the implant.

These include processing techniques such as crosslinking the materials.However, this typically does not create sufficient increased strength.In addition, the crosslinkers are usually cytotoxic, which adds thecomplexity of thoroughly rinsing the samples to remove excesscrosslinker. Other techniques have copolymerized the biomaterials with astiffer, usually synthesized, polymer. This greatly increases the costof manufacturing the material and can be hard to fabricate the materialinto a scaffold. In addition, scaffolds have been developed from arelatively stiff synthesized polymer with pore sizes in the range of afew hundreds of microns in diameter, which have then been coated with abiomaterial. This technique can provide the strength required, butresults in a mostly synthetic scaffold with minimal biomaterial that thecells cannot remodel. Once the biomaterials are degraded only thesynthetic polymer remains.

SUMMARY

The present disclosure provides new structures and methods to improvethe mechanical strength of a hybrid tissue scaffold of an engineeredtissue implant. The disclosure provides a hybrid tissue scaffoldcomprising a primary three-dimensional (base) scaffold which supports orotherwise carries a secondary three-dimensional scaffold, particularlywithin the pores thereof. More particularly, the porous primary scaffoldmay have a pore size in a range of between and including 0.50 mm to 5.0mm, while the secondary scaffold may have a relatively smaller poresize, such as in the range between and including 50 μm to 600 μm(microns).

The primary scaffold may be fabricated from a variety of polymermaterials and have different pore geometries to achieve the desiredstrength for a given application. The primary scaffold provides aframework or skeleton into which the secondary scaffold can beintroduced. In contrast to the primary scaffold, the secondary scaffoldmay particularly be formed of a natural biomaterial, such as a naturallyoccurring polymer. The naturally occurring polymer may be a protein(e.g. collagen, fibrin) or a carbohydrate (e.g. a polysaccharide such aschitosan, glycosaminoglycan).

The biomaterial, which may be in the form of a hydrogel, may be injectedinto the pores of the primary scaffold and reside therein. Thebiomaterial may be injected with or without seeding cells, and may beprocessed to provide a micro porous scaffold with designed pore size andgeometries.

In the foregoing manner, the primary scaffold will only occupy arelatively smaller volume of the hybrid scaffold or engineered tissueimplant, yet provide the needed strength of the implant for implantationwhile the biomaterial provides the appropriate environment for theseeding stem cells to proliferate.

The hybrid tissue scaffold or engineered tissue implant may acceleratethe wound healing process since they are able to: 1) cover the woundsite to keep the wound moist and to reduce the risk of infection; 2)reduce tissue regeneration time through the incorporation of functionaltissue developed in vitro; and 3) provide a source of stem cells to thewound sites for tissue regeneration.

In certain embodiments, a hybrid tissue scaffold may comprise a porousprimary scaffold having a plurality of pores, and a porous secondaryscaffold having a plurality of pores, wherein the secondary scaffoldresides in the pores of the primary scaffold to provide a hybridscaffold. The pores of the porous primary scaffold may have a pore sizein a range of 0.50 mm to 5.0 mm, and the pores of the porous secondaryscaffold have a pore size in a range of 50 μm to 600 μm. The primaryscaffold may provide 5% to 30% of a volume of the hybrid scaffold. Inother embodiments, the pores of the porous primary scaffold may have apore size in a range of 1 mm to 4 mm, and the pores of the poroussecondary scaffold may have a pore size in a range of 100 μm to 500 μm.

In certain embodiments, the porous primary scaffold may be formed of asynthetic polymer, and the synthetic polymer may be biodegradable in ahuman body. The synthetic polymer may be polyester, and may particularlyinclude polycaprolacone (PCL).

In certain embodiments, the secondary scaffold may be formed of abiomaterial which provides an environment to culture living cellstherein and promote attachment, migration, proliferation, and/orvascularization of the cells. The biomaterial may be formed of anaturally occurring polymer. The naturally occurring polymer may be aprotein (e.g. collagen, fibrin) and/or a carbohydrate. The secondaryporous scaffold may be in the form of a hydrogel or porous scaffold.

In certain embodiments, a plurality of living cells may be seeded to atleast one of the primary scaffold and the secondary scaffold. The cellsmay comprise at least one of endothelial, fibroblast and/or stem cells.

In certain embodiments, a method to provide a tissue scaffold comprisesforming a hybrid scaffold comprising a porous primary scaffold having aplurality of pores and a porous secondary scaffold having a plurality ofpores, wherein the secondary scaffold resides in the pores of theprimary scaffold to provide the hybrid scaffold. The pores of the porousprimary scaffold may have a pore size in a range of 0.50 mm to 5.0 mm,and the pores of the porous secondary scaffold may have a pore size in arange of 50 μm to 600 μm. The primary scaffold may provide 5% to 30% ofa volume of the hybrid scaffold.

In certain embodiments, the method may further comprise forming theporous primary scaffold with the plurality of pores and introducing theporous secondary scaffold into the pores of the porous primary scaffold.

In certain embodiment, the method may further comprise injecting theporous secondary scaffold into the pores of the porous primary scaffold.

In certain embodiments, the porous secondary scaffold is in the form ofa hydrogel, and the method may further comprise injecting the hydrogelinto the pores of the porous primary scaffold.

In certain embodiments, forming the hybrid scaffold may be performed insitu during a surgical procedure on a human body.

In certain embodiments, the method may further comprise fitting theporous primary scaffold to a tissue treatment site of a human bodybefore introducing the porous secondary scaffold into the pores of theporous primary scaffold.

FIGURES

The above-mentioned and other features of this disclosure, and themanner of attaining them, will become more apparent and betterunderstood by reference to the following description of embodimentsdescribed herein taken in conjunction with the accompanying drawings,wherein:

FIG. 1 is an image of a primary scaffold according to one embodiment ofthe present disclosure;

FIG. 2 is an image of a hybrid scaffold according to one embodiment ofthe present disclosure which makes use of the primary scaffold of FIG.1;

FIG. 3 is an image of the microstructure of PCL-collagen hybrid scaffoldof FIG. 2 imaged by an environmental electron scanning microscope(ESEM);

FIG. 4 is an image of live/dead staining of cells within the collagen ofa PCL-collagen hybrid scaffold; and

FIG. 5 is an image of live/dead staining of cells on the surface of thepolycaprolactone of a PCL-collagen hybrid scaffold.

DETAILED DESCRIPTION

It may be appreciated that the present disclosure is not limited in itsapplication to the details of construction and the arrangement ofcomponents set forth in the following description or illustrated in thedrawings. The invention(s) herein may be capable of other embodimentsand of being practiced or being carried out in various ways. Also, itmay be appreciated that the phraseology and terminology used herein isfor the purpose of description and should not be regarded as limiting assuch may be understood by one of skill in the art.

Referring now to figures, an exemplary primary scaffold according to thepresent invention is shown in FIG. 1 at reference character 10. Theprimary scaffold 10 provides a framework or skeleton into which asecondary scaffold may be introduced (e.g. injected or otherwise filled)into the plurality of pores 12 thereof. The pores of the primaryscaffold 10 may have a pore size in a range of between and including0.50 mm to 5.0 mm. More particularly, the pores of the primary scaffold10 may preferably have a pore size in a range of between and including1.0 mm to 4.0 mm. The primary scaffold 10 itself may have a porositylevel in a range of between and including 50% to 99%.

The primary scaffold 10 of FIG. 1 may preferably be made of abiodegradable polymer such as a polyester polymer includingpolycaprolactone (PCL) or poly(lactic-co-glycolic acid (PLGA). PCLreportedly has melting point of about 60° C. and a glass transitiontemperature of about −60° C. Reference to biodegradable may beunderstood herein as the ability to undergo enzymatic degradation ornonenzymatic hydroylysis and to provide non-toxic metabolites that maythen be eliminated by the body. It is therefore contemplated herein thatbiodegradable polymers suitable for preparation of the primary scaffold10 may also include polyurethane, poly (urethane urea),polyhydroxyalkanoates (PHAs), poly(1-lactic acid) (PLLA),polyanhydrides, polyfumerates, poly(n-isopropylacrylamide, orpolypeptides.

The fabrication of the primary scaffold herein may proceed by at leasttwo related methods. The first method may be generally understood asproviding a solution of the biodegradable polymer (polyester) that ismixed with insoluble inorganic salt particulate wherein the salt has asize range of 0.50 to 5.0 mm. This is then followed by relatively rapidcooling where the rate of cooling is such that the polyester (PCL) willprecipitate followed by evaporating the solvent before treatment withwater to dissolve the salt and provide a pore size range of 0.50 mm-5.0mm. Preferably, the rate of cooling is 1 degree Celsius per minute.Exemplary salts include NaCl, NaHCO₃, urea, and various sugars.

A preferred fabrication according to the first method noted above is tostart with a 10% (w/w) PCL solution made by dissolving PCL in organicsolvents, such as 65% (w/w) chloroform and methanol solution. The PCLsolution was then mixed with a salt that was previously sieved to therange of 0.85 mm to 1.40 mm. The solution was then placed in a freezerat −20° C. to provide PCL precipitation. The samples were then placed ina fume hood to allow the solvents to evaporate. Next, the samples areplaced in second solvent such as water to dissolve out the saltresulting in the formation of a porous PCL scaffold 10. As noted, suchporous PCL scaffold has a pore size range of 0.50-5.0 mm. See again,FIG. 1.

A second related method to fabricate the primary scaffold 10 may proceedby mixing a salt in a size range of 0.50 mm-5.0 mm with biodegradablepolymer particulate having a size less than or equal to 0.5 mm, or inthe range of 0.5 mm to 5.0 mm. The biodegradable polymer (e.g. PCL) maythen be heated at a temperature sufficient to sinter the PCL particlesor treated with solvent to form a continuous phase of the PCL around thesalt. Sintering may be understood as heating to a sufficient temperatureto join the PCL particles and the treatment with solvent is also suchthat the particles are joined to form the identified continuous phase.The salt is such that it again will not dissolve or melt, as the saltmay then again define the porosity to be achieved when the salt isultimately removed from the continuous phase of biodegradable polymer(PCL). That is, the salt, present at a size of 0.50 mm to 5.0 mm, isdissolved to obtain the porous PCL scaffold 10. The pore size of the PCLscaffold may again be in the range of 0.50 mm to 5.0 mm, and preferablyin the range of 1.0 mm to 4.0 mm (due to use of correspondingly lowersalt size). The PCL scaffold 10 then once again serves as the skeletonof the hybrid tissue scaffold/engineered tissue implant disclosedherein, and provides a framework for the hybrid tissue scaffold/implantto withstand the forces during in vivo implantations. As noted above,the PCL scaffold is configured such that it will amount to 5.0% to 30.0%of the hybrid scaffold volume, more preferably in the range of 10.0% to20.0% by volume, and more preferably, 14.0% to 16.0% by volume.

Once the primary scaffold 10 is fabricated, a porous secondary scaffold20 may be introduced into the pores 12 of the primary scaffold 10.Referring now to FIG. 2, a secondary scaffold 20 is shown residing in,such as by being injected into the pores in the primary scaffold 10. Thesecondary scaffold 20 is preferably formed of a biomaterial (any matter,surface, or construct that interacts with biological systems of a humanbody) and which provides an environment to culture living seeded cellsand promote cell attachment, migration, proliferation, and/orvascularization. To facilitate injection and biological interaction, thesecondary scaffold 20 may particularly be in the form of a hydrogel. Ahydrogel herein may be understood as a network of polymer chains thatare hydrophilic in which water is the dispersion medium.

The pores of the secondary scaffold 20 may have a pore size in a rangeof between and including 50 μm to 600 μm. More particularly, the poresof the secondary scaffold 20 may have a pore size in the range of 200 μmto 500 μm. The secondary scaffold 20 may have a porosity level in arange of between and including 50% to 99% by volume.

The biomaterial may be formed of a naturally occurring polymer which isa protein (e.g. collagen, fibrin) and/or a carbohydrate e.g. apolysaccharide such as chitosan, glycosaminoglycan. FIG. 2 shows asecondary scaffold 20 of collagen introduced to and residing in thepores 12 of primary scaffold 10, the combination of which provides aPCL-collagen hybrid scaffold 30.

FIG. 3 shows the microstructure of PCL-collagen hybrid scaffold 30imaged by an environmental electron scanning microscope (ESEM). Arrow Aillustrates the location of the PCL primary scaffold 10, while arrow Billustrates the location of the collagen secondary scaffold 30. Thecollagen scaffold 30 has interconnected pores with an average diameterof 250 μm within the collagen material and the porosity was about 70%.

As noted, the primary scaffold herein may provide 5.0% to 30.0% of thehybrid scaffold volume. In this manner, the primary scaffold 10 mayprovide the needed strength of the hybrid scaffold/implant forimplantation while the filler biomaterial of the secondary scaffold 20provides the needed environment for seeded stem cells to proliferate.While the primary PCL scaffold 10 therefore represents only a portion ofthe volume of the PCL-collagen hybrid scaffold 30, the PCL scaffold 10may now unexpectedly enhance the mechanical stiffness of thePCL-collagen hybrid scaffold 30 while preserving the ability of thescaffold to provide the correct material environment for cellproliferation.

More specifically, it has been found that the PCL-collagen hybridscaffold 30 exhibits a Young's modulus in the range of 0.1 MPA to 100GPa, more preferably, 1.80 to 2.2 MPa. Young's modulus of the hybridscaffold is measured by unconfined compression. By comparison, it hasbeen found that scaffolds which rely upon only crosslinked collagen(where crosslinking was employed to increase the modulus value andovercome the problem of relatively poor mechanical strength), indicateYoung's modulus value of about 23 kPa. As can therefore be appreciated,the present hybrid scaffold provides magnitudes of order improvement inmechanical property characteristics, such as Young's modulus, therebyachieving the goal of strength improvement in the hybrid scaffoldwithout the need to crosslink and/or extraction of chemical crosslinkingagents. In addition, the present hybrid skeleton approach allows formechanical property improvement without the need to crosslink thebiomaterial. A scaffold is nonetheless produced that withstands loadingforces during implantation where it may be press-fit into a selectedlocation.

Additional embodiments of the hybrid scaffold 30 include using fibrin asthe secondary scaffold 20 to be introduced into the pores of the primaryscaffold 10 instead of collagen. Both the PCL-collagen hybrid scaffold30 and the PCL-fibrin hybrid scaffold 30 have been found to successfullyhouse seeded cells, such as stem, endothelial, and fibroblast cells, andallow them to create extracellular matrix and remodel the material in anin vitro culture. FIG. 4 shows live/dead staining of cells within thecollagen of the PCL-collagen hybrid scaffold 30. FIG. 5 shows live/deadstaining of cells on the surface of the polycaprolactone of thePCL-collagen hybrid scaffold 30.

Thus, the foregoing technique may also provide enhanced versatility ofapplications. The pores of the primary scaffold may be injected orotherwise filled with the biomaterial and processed to form the hybridtissue scaffold/engineered implant prior to use thereof. The hybridtissue scaffold therefore relies only upon the presence of the primaryscaffold of the indicated polymers and secondary scaffold material asdisclosed herein and avoids dependence on the use of any othercomponents (e.g. crosslinking agents in the secondary scaffold or theuse of copolymer structure in the secondary scaffold biomaterial). Thehybrid tissue scaffold also does not rely upon the presence of glass.Accordingly, the present disclosure provides transplantable hybridtissue scaffolds/implants that can be vascularized or even pre-developedinto functional tissue in vitro and then transplanted in vivo asengineered tissue substitute at a tissue treatment site. The hybridtissue scaffold/engineered implant may be configured to connect withhost blood vessels in vivo to supply oxygen and nutrition to cellsthereof immediately after the implantation.

In addition, as alluded to above, the bare primary scaffold can beshapeable as to conform to the anatomical shape of a wound or othertissue treatment site (e.g. press fit and/or cut to fit) and then thebiomaterial can be injected into the primary scaffold in situ in aminimally invasive procedure. The biomaterial is configured to integrateinto surrounding host tissue to achieve wound healing. The hybrid tissuescaffold or engineered tissue implant may comprise a same or similartissue type as the lost tissue at the tissue treatment site. The tissuescaffold construct/engineered tissue implant may be developed intofunctional tissue types such as muscle, bone, cartilage, and epithelialin vitro. Further, the hybrid tissue scaffold may be loaded with drugsor growth factors to better control the integration into the surroundingtissue.

While a preferred embodiment of the present invention(s) has beendescribed, it should be understood that various changes, adaptations andmodifications can be made therein without departing from the spirit ofthe invention(s) and the scope of the appended claims. The scope of theinvention(s) should, therefore, be determined not with reference to theabove description, but instead should be determined with reference tothe appended claims along with their full scope of equivalents.Furthermore, it should be understood that the appended claims do notnecessarily comprise the broadest scope of the invention(s) which theapplicant is entitled to claim, or the only manner(s) in which theinvention(s) may be claimed, or that all recited features are necessary.

What is claimed is:
 1. A method of forming a hybrid tissue scaffold comprising: forming a solution of biodegradable polymer in the presence of an inorganic salt wherein the inorganic salt is water soluble and has a particulate size in a range of 0.50 to 5.0 mm; cooling said solution to precipitate said biodegradable polymer followed by treatment with water to dissolve said inorganic salt wherein the biodegradable polymer is recovered as a porous primary scaffold having a pore size in a range of 1 mm to 4 mm; introducing into said primary scaffold a biomaterial comprising a hydrogel including living cells and forming a secondary scaffold from said biomaterial including living cells, wherein the secondary scaffold resides in the pores of the primary scaffold to provide a hybrid scaffold; wherein said biomaterial provides an environment to culture the living cells and said biomaterial has a pore size of 50 μm to 600 μm; and wherein said primary scaffold is present at 5.0% to 30.0% by volume of said hybrid scaffold and said hybrid scaffold has a Young's modulus of 0.1 MPA to 100 GPa.
 2. The method of claim 1 wherein said biodegradable polymer comprises a polyester polymer.
 3. The method of claim 1 wherein said biomaterial does not contain any crosslinking.
 4. The method of claim 1 wherein said biodegradable polymer is recovered as a porous primary scaffold having a pore size in the range of 1.0 mm to 4.0 mm.
 5. The method of claim 1 wherein the primary scaffold is present at 10.0% to 20.0% by volume.
 6. The method of claim 1 wherein said biomaterial has a porosity of 50% to 99% by volume.
 7. The method of claim 1 wherein said biomaterial further comprises at least one of a protein and a carbohydrate.
 8. (canceled)
 9. A method of forming a hybrid tissue scaffold comprising: mixing biodegradable polymer particulate size in a range of 0.50 mm to 5.0 mm with an inorganic salt having a particulate size in a range of 0.5 mm to 5.0 mm; sintering or treating said biodegradable polymer with solvent to form a continuous phase of the biodegradable polymer around said salt; dissolving said inorganic salt wherein said biodegradable polymer is recovered as a porous primary scaffold having a pore size in a range of 1 mm to 4 mm; introducing into said primary scaffold a biomaterial comprising a hydrogel including living cells and forming a secondary scaffold from said biomaterial including living cells, wherein the secondary scaffold resides in the pores of the primary scaffold to provide a hybrid scaffold; wherein said biomaterial provides an environment to culture the living cells and said biomaterial has a pore size of 50 μm to 600 μm; and wherein said primary scaffold is present at 5.0% to 30.0% by volume of said hybrid scaffold and said hybrid scaffold has a Young's modulus of 0.1 MPA to 100 GPa.
 10. The method of claim 9 wherein said biodegradable polymer comprises a polyester.
 11. The method of claim 9 wherein said biomaterial does not contain any crosslinking.
 12. The method of claim 9 wherein said biodegradable polymer is recovered as a porous primary scaffold having a pore size in the range of 1.0 mm to 4.0 mm.
 13. The method of claim 9 wherein the primary scaffold is present at 10.0% to 20.0% by volume.
 14. The method of claim 9 wherein said biomaterial has a porosity of 50% to 90% by volume.
 15. The method of claim 9 wherein said biomaterial further comprises at least one of a protein and a carbohydrate.
 16. (canceled)
 17. A hybrid tissue scaffold comprising: a porous primary scaffold of biodegradable polymer having a plurality of pores having a pore size range of 0.50 mm to 5.0 mm; a porous secondary scaffold of biomaterial having a plurality of pores having a pore size range of 50 μm to 600 μm; wherein the primary scaffold provides 5% to 30% of a volume of the hybrid scaffold and said hybrid scaffold has a Young's modulus of 0.1 MPA to 100 GPa.
 18. The tissue scaffold of claim 17 wherein: the pores of the porous primary scaffold have a pore size in a range of 1.0 mm to 4.0 mm.
 19. The tissue scaffold of claim 17 wherein: the pores of the porous secondary scaffold have a pore size in a range of 200 μm to 500 μm.
 20. The tissue scaffold of claim 17 wherein: the porous primary biodegradable scaffold is formed of a synthetic polymer.
 21. The tissue scaffold of claim 20 wherein: the synthetic polymer is a polyester.
 22. The tissue scaffold of claim 17 wherein: the biomaterial is formed of a naturally occurring polymer.
 23. The tissue scaffold of claim 22 wherein: the naturally occurring polymer is a protein.
 24. The tissue scaffold of claim 23 wherein: the protein is collagen.
 25. The tissue scaffold of claim 23 wherein: the protein is fibrin.
 26. The tissue scaffold of claim 22 wherein: the naturally occurring polymer is a carbohydrate.
 27. The tissue scaffold of claim 17 wherein: the secondary porous scaffold is a hydrogel.
 28. The tissue scaffold of claim 17 further comprising: a plurality of living cells seeded to at least the secondary scaffold.
 29. The tissue scaffold of claim 28 wherein: the cells comprise at least one of endothelial, fibroblast and stem cells. 